Immunoglobulins stabilized with a chelator of copper ions

ABSTRACT

A stabilized immunoglobulin composition comprises at least one immunoglobulin together with a stabilizing amount of a chelator of copper ions such as EDTA or citrate. Preferably the immunoglobulin is an antibody, for example, a recombinant CDR-grafted antibody. A process for enhancing the stability of an immunoglobulin comprises subjecting the immunoglobulin to a purification procedure capable of removing copper ions therefrom. Preferably, the immunoglobulin is rendered substantially free from detectable copper ions as determined, for example, by atomic absorption spectroscopy.

This application was filed under 35 U.S.C. 371 as the national stage ofPCT international application no. PCTGB201970, filed Oct. 27, 1992.

The present invention relates to the stabilisation of immunoglobulinsagainst degradation, in particular on storage and processing prior touse.

Antibodies or immunoglobulins are proteinaceous bifunctional molecules.One part, which is highly variable between different antibodies, isresponsible for binding to an antigen, for example the many differentinfectious agents that the body may encounter, whilst the second,constant, part is responsible for binding to the Fc receptors of cellsand also activates complement. In this way, antibodies represent a vitalcomponent of the immune response of mammals in destroying foreignmicroorganisms and viruses.

The immunisation of an animal with an antigen results in the productionof different antibodies with different specificities and affinities. Anantiserum obtained from the immunised animal will, therefore, beheterogeneous and contain a pool of antibodies produced by manydifferent lymphocyte clones. Antibodies thus obtained are referred to aspolyclonal antibodies and this polyclonal nature has been a majordrawback in the use of antibodies in diagnostic assays and intherapeutic applications.

A major step forward occurred in 1975 when Kohler and Milstein (Nature,1975, 256, 495-497) reported the successful fusion of spleen cells frommice immunized with an antigen with cells of a murine myeloma line. Theresulting hybrid cells, termed hybridomas, have the properties ofantibody production derived from spleen cells and of continuous growthderived from the myeloma cells. Each hybridoma synthesizes and secretesa single antibody to a particular determinant of the original antigen.To ensure that all cells in a culture are identical, i.e. that theycontain the genetic information required for the synthesis of a uniqueantibody species, the hybridomas resulting from cell fusion are clonedand subcloned. In this way, the cloned hybridomas produce homogeneous ormonoclonal antibodies.

The advantages of hybridoma technology are profound. Because manyhybrids arising from each spleen are screened for their potential toproduce antibodies to the antigen of interest and only a few areselected, it is possible to immunize with impure antigens and yet obtainspecific antibodies. The immortality of the cell line assures that anunlimited supply of a homogeneous, well-characterised antibody isavailable for use in a variety of applications including in particulardiagnosis and immunotherapy of pathological disorders. Unfortunately,the usefulness of such monoclonal antibodies in a clinical setting canbe severely hampered by the development of human anti-mouseantibodies--an anti-globulin response--which may interfere with therapyor cause allergic or immune complex hypersensitivity. This has led tothe development of humanised antibodies.

An antibody molecule is composed of two light chains and two heavychains that are held together by interchain disulphide bonds. Each lightchain is linked to a heavy chain by disulphide bonds and the two heavychains are linked to each other by disulphide bonds. Each heavy chainhas at one end a variable domain followed by a number of constantdomains, and each light chain has a variable domain at one end and aconstant domain at the other end. The light chain variable domain isaligned with the variable domain of the heavy chain. The light chainconstant domain is aligned with the first constant domain of the heavychain. The remaining constant domains of the heavy chains are alignedwith each other. The constant domains in the light and heavy chains arenot involved directly in binding the antibody to the antigen.

The variable domains of each pair of light and heavy chains form theantigen binding site. They have the same general structure with eachdomain comprising a framework of four regions, whose sequences arerelatively conserved, connected by three complementarity determiningregions (CDRs). The four framework regions largely adopt a beta-sheetconformation and the CDRs form loops connecting, and in some casescomprising part of, the beta-sheet structure. The CDRs are held in closeproximity by the framework regions and, with the CDRs from the otherdomain, contribute to the formation of the antigen binding site.

In the use of murine monoclonal antibodies, the induction of an humananti-mouse antibody response is due to the murine origin of the constantdomains and four framework regions. This problem has therefore beenaddressed by the development of modified antibodies of two basic types.The first type, referred to as chimeric antibodies, is where the murineconstant domains only are replaced by equivalent domains of human origin(Morrison et al, P.N.A.S., 1984, 81, 6851-6855; Boulianne et al, Nature,1985, 314, 268-270; and Neuberger et al, Nature, 1985, 314, 268-270).The second type is where the murine constant domains and the murineframework regions are all replaced by equivalent domains and regions ofhuman origin. This second type of modified antibody is referred to as ahumanised or CDR-grafted antibody (Jones et al, Nature, 1986, 321,522-525; and Riechmann et al, Nature, 1988, 332, 323-327).

To generate sufficient quantities of antibody for full clinicalinvestigation, it is desirable to utilize an efficient recombinantexpression system. Since myeloma cells represent a natural hostspecialized for antibody production and secretion, cell lines derivedfrom these have been used for the expression of recombinant antibodies.Often, complex vector design, based around immunoglobulin generegulatory elements, is required, and final expression levels have beenreported which are highly variable (Winter et al, Nature, 1988, 332,323-327; Weidle et al, Gene, 1987, 60, 205-216; Nakatani et al,Bio/Technology, 1989, 7, 805-810; Gillies et al, Bio/Technology, 989, 7,799-804).

Other types of expression systems which have been proposed forantibodies include immortalised human B cells (Rice et al, Proc. Natl.Acad. Sci. USA, (1982) 79 862-7865), however yields are generally lowand it is difficult to establish stable cell lines. E. coli has beenused to express F_(v) fragments (Skerra & Plukthun, Science, (1988) 240,1038-1041) or single chain antigen binding molecules (Bird et al,Science, (1988) 242, 423-426) but entire immunoglobulins have so far notbeen produced in the system. Antibodies have, however, been successfullyproduced in mammalian cell expression systems which are already knownfor the production of recombinant proteins such as Chinese hamster ovary(CHO) cells.

In the production of purified antibodies whether for therapeutic ordiagnostic use, it is important that the antibody is sufficiently stableon storage and various chemical entities may have an adverse effect onthe stability of the antibody. The present invention is based on thesurprising discovery that trace amounts of copper (Cu⁺⁺) have adestabilising effect on immunoglobulin molecules on storage and thatthis effect can be eliminated by formulating the immunoglobulin moleculewith a suitable chelator of copper ions.

It has also surprisingly been found that the presence of a chelator ofcopper ions may have a stabilising effect on the immunoglobulin moleculeeven when the immunoglobulin does not contain amounts of copper whichare detectable by conventional techniques such as atomic absorptionspectroscopy. Whilst not wishing to be bound by any particular theory,it may be that the presence of copper ions in amounts below thedetection limits of techniques such as atomic absorption spectroscopystill has a destabilishing effect on the immunoglobulin molecule whichcan be eliminated by the addition of a suitable chelating agent.

The present invention provides a stabilised immunoglobulin compositioncomprising at least one immunoglobulin together with a stabilisingamount of a chelator of copper ions.

The invention also provides the use of a chelator of copper ions tostabilise an immunoglobulin against degradation on storage, for exampledegradation resulting from the effect of copper ions.

The fact that trace amounts of copper ions have a destabilising effecton immunoglobulins means that there may be an advantage in terms ofstability in ensuring that immuncglobulins contain the minimum possibleamount of copper ions. According to a further aspect the presentinvention provides a purified immunoglobulin substantially free fromcopper ions. In particular the invention provides an immunoglobulin inwhich no copper can be detected by the use of conventional techniquessuch as atomic absorption spectroscopy.

The invention also provides a process for enhancing the stability of animmunoglobulin which comprises subjecting the immunoglobulin to apurification procedure capable of removing copper ions therefrom. Inparticular the procedure should be such that no copper can be detectedin the immunoglobulin by the use of conventional procedures such asatomic absorption spectroscopy. Copper can be removed fromimmunoglobulins by conventional procedures known in the field of proteinpurification such as dialysis versus potassium cyanide containingphosphate buffer followed by gel filtration to remove copper as coppercyanide (see for example Baker and Hultquist, J. Biol. Chem., 253,844-845 (1978)).

The present invention is applicable to the stabilisation ofimmunoglobulins of all classes, i.e IgM, IgG, IgA, IgE and IgD, and italso extends to the stabilisation of Fab fragments and bispecificantibodies. The invention is preferably applied to the stabilisation ofinmunoglobulins of the class IgG, which includes the sub-classes IgG₁,IgG_(2A), IgG_(2B), IgG₃ and IgG.sub. 4. The invention is morepreferably applied to the stabilisation of immunoglobulins of the classIgG₁.

The invention finds particular application in the stabilisation ofrecombinant antibodies, most particularly chimeric antibodies orhumanised (CDR-grafted) antibodies. Particular examples of these includechimeric or humanised antibodies against CD2, CD3, CD4, CD5, CD7, CD8,CD11a,b, CD18, CD19, CD25, CD33, CD54 and especially humanisedantibodies against the CDw52 antigen, such as CAMPATH-1H (CAMPATH is aTrade Mark of the Wellcome group of companies). Further examples includechimetic or humanised antibodies against various tumour cell markerantigens.

The immunoglobulin will generally be formulated with the metal ionchelating agent at an early stage, for example during or immediatelyfollowing purification. The production procedure for an immunoglobulinwill generally involve purification by means of chromatography and/orgel filtration columns. The chelating agent can be added at anyconvenient stage of the purification procedure, for example at the stageof the final column, so that the chelating agent remains in theimmunoglobulin at the end of the purification procedure. Alternatively,the chelating agent may be added at a suitable stage followingpurification. In the case of a lyophilised immunoglobulin the chelatingagent will generally be added prior to lyophilisation.

The level at which the chelating agent is added to the immunoglobulinwill be such as to ensure that any copper present is bound by thechelating agent and thus rendered ineffective in destabilising theimmunoglobulin. The invention is applicable irrespective of the intendedend use of the immunoglobulin although the chelating agent which is usedshould be chosen in such a way that it will not have an adverse effecton the intended end use of the immunoglobulin. For example in the caseof antibodies intended for therapeutic use, the chelating agent shouldnot show any toxic effects at the level in which it will be present.

A particularly preferred metal ion chelating agent is ethylenediaminetetraacetic acid (EDTA) which may typically be added to theimmunoglobulin at levels of 0.05 mM to 5 mM, preferably 0.1 mM to 3 mM.A level of 0.1 mM EDTA will often be sufficient to stabilise animmunoglobulin but levels up to 2 mM or higher do not present anyproblem physiologically in the case of an immunoglobulin intended foradministration to humans. An alternative metal ion chelating agent iscitrate ion, preferably used in the form of an alkali metal citrate,e.g. sodium citrate.

Immunoglobulins intended for therapeutic use will generally beadministered to the patient in the form of a pharmaceutical formulation.Such formulations preferably include, in addition to the immunoglobulin,a physiologically acceptable carrier or diluent, possibly in admixturewith one or more other agents such as other immunoglobulins or drugs,such as an antibiotic. Suitable carriers include, but are not limitedto, physiologic saline, phosphate buffered saline, phosphate bufferedsaline glucose and buffered saline. Alternatively the immunoglobulin maybe lyophilised (freeze dried) and reconstituted for use when needed bythe addition of an aqueous buffered solution as described above. Routesof administration are routinely parenteral, including intravenous,intramuscular, subcutaneous and intraperitoneal injection or delivery.The chelating agent may be incorporated into any type of immunoglobulinformulation intended either for storage and distribution or ultimateuse. The pharmaceutical formulation will generally contain, or in thecase of a lyophilised preparation will be reconstituted to contain, aneffective therapeutic dose of the immunoglobulin per unit dose. In thecase of the humanised antibody CAMPATH-1H, liquid formulations orreconstituted lyophilised formulations preferably contain 0.5 to 20mg/ml of the antibody, preferably 2 mg/ml or 10 mg/ml.

The invention is illustrated by the following examples:

EXAMPLE 1

The effect of various additives on the stability of a recombinantantibody was studied at 37° C. The antibody was CAMPATH 1H, a humanisedantibody against the CDw52 antigen (Riechmann et al, Nature, 332,323-327 (1988)), which had been produced by expression in a recombinantCHO cell line transformed with DNA encoding the heavy and light chainsof the antibody molecule. The antibody was extracted from the cellculture medium and purified and was then stored as a solution (1 mg/ml)in phosphate buffered saline at +4° C.

Vials containing 0.5 ml of the solution of CAMPATH 1H referred to abovetogether with the additive specified were incubated at +37° C. for 4weeks under sterile conditions. At the end of this period the sampleswere analysed by size exclusion HPLC, the stability of the sample beingassessed by the extent of the formation of "peak C" (a peak formed bythe major degradation product of the antibody which has a molecularweight of about 50K) based on the total eluted protein. The results areset out in the following Table 1.

                  TABLE 1                                                         ______________________________________                                        ADDITIVE           % PEAK C                                                   ______________________________________                                        None               12%                                                        None (storage at +4° C.)                                                                   2%                                                        Cu.sup.++  (10 ppm)                                                                              28%                                                        EDTA (2 mM)        <1%                                                        1,10-phenanthroline (10 mM)                                                                       3%                                                        ______________________________________                                    

The copper was added as CuCl₂.2H₂ O and the 1,10-phenanthroline as asolution in water containing 2% (v/v) ethanol.

These results demonstrate that copper enhances the degree of degradationof the antibody relative to the control. The addition of EDTA virtuallyeliminates degradation whilst the other metal ion chelator1,10-phenanthroline reduces degradation to a considerable extent.

EXAMPLE 2

This example also used CAMPATH 1H produced in CHO cells of the typereferred to in Example 1 (11.3 mg/ml in phosphate buffered saline) andthe batch having been measured as containing 0.04 μg Cu² + /ml. In thisand following examples, the copper content of antibody samples wasmeasured by atomic absorption spectroscopy using a Philips PU9400Xatomic absorption spectrophotometer. The detection limit of this methodwas about 0.03 μg Cu/ml so that samples stated to have "no detectablecopper" contain less than 0.03 μg Cu/ml. Samples of this CAMPATH-H(anti-CDw52 antibody) 1H were diluted to 1 mg/ml in phosphate bufferedsaline and dialysed exhaustively versus 0.2M sodium phosphate buffer atpH 6.0, pH 6.4 and pH 6.8. CAMPATH 1H previously having been determinedto be most stable against degradation by heat at about pH 6. Thefollowing was added to 300 μl samples at each pH:

(i) 30 μl 10 mM CuCl₂.2H₂ O in water;

(ii) 30 μl 10 mM EDTA in water;

(iii) 30 μl buffer;

and the samples incubated at 62° C. for 24 hours. 50 μl aliquots wereanalysed as described in Example 1 with degradation being assessed bysize exclusion chromatography and measured as the extent of formation of"Peak C" based on the total eluted protein.

The results for % Peak C are given in Table 2 below:

                  TABLE 2                                                         ______________________________________                                        % Peak C                                                                      pH      Cu            EDTA    Buffer                                          ______________________________________                                        6.0     1.75          0.38    0.69                                            6.4     2.94          0.34    0.72                                            6.8     5.31          0.51    1.12                                            ______________________________________                                    

The results indicate that as pH increases, the effect of copper on thedegradation of CAMPATH 1H increases. In the absence of added copper anincrease in % Peak C is also seen with increasing pH. In the presence ofEDTA the degradation of CAMPATH 1H is suppressed.

EXAMPLE 3

This example used two different batches of CAMPATH 1H produced in CHOcells of the type referred to in Example 1 (10 mg/ml in phosphatebuffered saline): Batch 1 contained no detectable Cu²⁺ as determined byatomic absorption spectroscopy and Batch 2 contained 0.04 μg Cu²⁺ /ml.Samples of both batches were diluted to 1 mg/ml in phosphate bufferedsaline and dialysed extensively for 24 hours at +4° C. against 50 mMammonium hydrogen carbonate and 1 mM EDTA was add to the Batch 2 toeliminate any effect of the copper. 200 μl aliquots of both batches wereincubated for 24 hours at 4°, 10°, 20°, 30°, 40°, 50° and 62° C. anddegradation was assessed as described in Example 1 by size exclusionchromatography and measured as the extent of formation of "Peak C" basedon the total eluted protein.

The results for % Peak C are given in Table 3 below:

                  TABLE 3                                                         ______________________________________                                                     % Peak C                                                         Temperature    Batch 1 Batch 2 + EDTA                                         ______________________________________                                         4° C.  0       0                                                      10° C.  0       0                                                      20° C.  0       0                                                      30° C.  0.47    0                                                      40° C.  2.71    0                                                      50° C.  60.1    0                                                      62° C.  72.36   1.12                                                   ______________________________________                                    

Although no detectable Cu.sup. 2+ was found in Batch 1, some degradationwas apparent on incubation at 30° and 40° C. with extensive degradationat 50° and 62° C. In the case of Batch 2 which contained detectableCu²⁺, minimal degradation was seen even at elevated temperature in thepresence of EDTA. These results suggest the possibility thatsubdetectable levels of Cu²⁺ may accelerate the degradation of CAMPATH1H.

EXAMPLE 4

The results of Example 1 were confirmed by a timed incubation at 62° C.over a period of 24 hours using the same CAMPATH 1H antibody produced inCHO cells. The batch used was determined to contain. 0.03 μg Cu²⁺ /ml byatomic absorption spectroscopy and 3 ml of this batch containing 3.7mg/ml CAMPATH 1H in phosphate buffered saline was dialysed at +4° C. for24 hours against 3×2 liters 50 mM ammonium hydrogen carbonate. 100 μlaliquots were incubated at 62° C. with the following additions:

(i) 5 μl 0.01M EDTA in water+10 μl 0.1M CuCl₂.2H₂ O in water;

(ii) 5 μl 0.01M in water;

(iii) none.

The amount of EDTA added should have been sufficient to chelate anyresidual transition metal ions in the antibody but not sufficient tochelate the copper which is added in Sample (i).

50 μl samples were withdrawn for analysis at the following times: 0, 1,2, 3, 4, 5 and 24 hours. The samples were analysed as in Example 1 bysize exclusion HPLC with the extent of formation of Peak C again beingtaken as a measure of the extent to which the antibody had beendegraded. The results are shown in the following Table 4:

                  TABLE 4                                                         ______________________________________                                        Time    % Peak C                                                              hours   EDTA + Cu       EDTA    None                                          ______________________________________                                        0       0               0       0                                             1       2.49            0       1.13                                          2       9.20            0       1.82                                          3       39.24           0       3.27                                          4       44.83           0       5.13                                          5       49.42           0       6.89                                          24      100             2.25    22.12                                         ______________________________________                                    

EXAMPLE 5

This example also used CAMPATH 1H produced in CHO cells of the typereferred to in Example 1 (10.0 mg/ml in phosphate buffered saline) andthe batch having no detectable copper as measured by atomic absorptionspectroscopy. A sample of this CAMPATH-1H (anti-CDw52 antibody) wasdialysed at +4° C. versus 50 mM ammonium hydrogen carbonate and 100 μlaliquots were incubated at 62° C. for 24 hours with 10 μl of increasingconcentrations of CuCl₂.2H₂ O in water. The samples were analysed as inExample 1 by size exclusion HPLC with the extent of formation of "PeakC" again being taken as a measure of the extent to which the antibodyhad been degraded. The results are shown in the following Table 5:

                  TABLE 5                                                         ______________________________________                                        nMoles Cu per                                                                 nMole CAMPATH 1H % Peak C                                                     ______________________________________                                        0                1.61                                                         0.018            8.09                                                         0.037            11.41                                                        0.074            13.61                                                        0.145            17.59                                                        0.293            22.84                                                        ______________________________________                                    

The extent of degradation was found to increase with increasing molarratio of Cu²⁺ /CAMPATH 1H. At ratios above 0.3 (data not shown),aggregation was seen with lower recovery of total protein.

EXAMPLE 6

This example also used CAMPATH 1H produced in CHO cells of the typereferred to in Example 1 (1.0 mg/ml in phosphate buffered saline), thebatch having been found to contain 0.19 μg Cu²⁺ /ml as measured byatomic absorption spectroscopy. The sample thus had a high coppercontent (copper/CAMPATH 1H molar ratio 449 pMol Cu²⁺ /nMol CAMPATH 1H)and early stability studies showed that this batch was subject tosubstantial degradation on storage at 37° C.

The effect of incubation of this sample for up to four weeks at 37° C.with and without the presence of 2 mM EDTA is shown below in Table 6.The samples were analysed as in Example 1 by size exclusion HPLC withthe extent of formation of "Peak C" again being taken as a measure ofthe extent to which the antibody had been degraded.

                  TABLE 6                                                         ______________________________________                                        Time           % Peak C                                                       (weeks)        2 mM EDTA  No EDTA                                             ______________________________________                                        1              0.72       2.86                                                2              1.26       6.59                                                3              1.24       9.24                                                4              1.44       10.18                                               4 at +4° C.                                                                           0.95       1.02                                                ______________________________________                                    

2 mM EDTA substantially decreases the decomposition of the CAMPATH 1Hbut does not totally inhibit it.

A sample of the same CAMPATH-1H (anti-CDw52 antibody) was dialysed at+4° C. versus 50 mM ammonium hydrogen carbonate and 100 μl aliquots wereincubated at 62° C. for 24 hours with varying concentrations of EDTA.Again the samples were analysed as in Example 1 by size exclusion HPLCwith the extent of formation of "Peak C" being taken as a measure of theextent to which the antibody had been degraded. The results of twoseparate experiments are shown in Tables 7 and 8 below:

                  TABLE 7                                                         ______________________________________                                               mM EDTA % Peak C                                                       ______________________________________                                               0       6.86                                                                    0.1   1.03                                                                  1       1.38                                                                  2       1.12                                                                  3       1.26                                                                  4       1.04                                                                  10      1.20                                                           ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                               mM EDTA % Peak C                                                       ______________________________________                                               0       7.47                                                                  0.0001  8.43                                                                  0.001   7.28                                                                  0.01    1.83                                                                  0.04    1.68                                                                  0.1     1.63                                                           ______________________________________                                    

These results show that as little as 0.01 mM EDTA effectively inhibitsdecomposition of CAMPATH 1H.

EXAMPLE 7

The effect of Cu²⁺ and of EDTA on the decomposition of variousantibodies is shown in Table 9 below. All samples were incubated at 4°C. and at 62° C. for 24 hours in the absence of any additives and at 62°C. for 24 hours in the presence of either Cu²⁺ (1 mM CuCl₂.2H₂ O+0.5 mMEDTA) or EDTA (1 mM EDTA).

                  TABLE 9                                                         ______________________________________                                               % Peak C                                                                        4° C.                                                                           62° C.                                                                            62° C.                                                                       62° C.                              Antibody No EDTA  No EDTA    + Cu.sup.2+                                                                         + EDTA                                     ______________________________________                                        IgG1     0.54     1.58       5.59  1.1                                        C1H      0        2.49       27.98 0                                          CD4      0.4      1.91       21.52 1.84                                       IgG2     0        1.81       3.77  0                                          ______________________________________                                         IgGl = mouse monoclonal IgG.sub.1 antibody, 1 mg/ml in phosphate buffered     saline;                                                                       C1H = CAMPATH 1H of the type described in Example 1, 1 mg/ml in phosphate     buffered saline;                                                              CD4 = Humanised antiCD4 monoclonal antibody having the same framework         region as CAMPATH 1H and produced in CHO cells, 1 mg/ml in phosphate          buffered saline;                                                              IgG2 = Mouse IgG.sub.2 monoclonal antibody I4139 commercially available       from Sigma, supplied lyophilised from phosphate buffer and redissolved        with water to 1 mg/ml.                                                   

All samples show little or no decomposition at 4° C. whereas there issome decomposition at 62° C. which is increased by varying degrees bythe presence of copper. Decomposition at 62° C. is suppressed by EDTA.

EXAMPLE 8

A comparison between the effect of 2 mM EDTA in phosphate bufferedsaline (pH 7.2) and 50 mM citrate (pH 6.0) on the stability ofCampath-1H was carried out at various levels of copper. Campath-1Hproduced in CHO cells of the type referred to in Example 1, the batchhaving no detectable copper as measured by atomic absorptionspectroscopy, was diluted 1 in 10 by volume with phosphate bufferedsaline. 1 ml aliquots were dialysed against 1 liter of the followingbuffers:

(i) phosphate buffered saline, pH 7.2;

(ii) 2 mM EDTA in phosphate buffered saline, pH 7.2;

(iii) 50 mM sodium citrate, pH 6.0.

Dialysis was carried out at 4° C. with three changes over 16 hours.Protein concentration was then determined for the three samples byscanning between 340 and 200 nm using a buffer blank and taking theextinction coefficient A₂₈₀ (0.1%) as 1.32. Protein concentrations of:

(i) 1.32 mg/ml

(ii) 1.20 mg/ml

(iii) 1.27 mg/ml

were determined.

200 μl aliquots of the antibody in the above buffers were then incubatedwith increasing concentrations of CuCl₂.2H₂ O (up to 20 mM) at 62° C.for 24 hours (62° C. being the optimal temperature for copper-inducedcleavage of CAMPATH-1H (anti-CDw52 antibody) Samples (50 μl aliquots)were then analysed by size exclusion HPLC in the manner described inExample 1 and the various fractions integrated by cutting and weighingchromatograms of the A₂₈₀ -absorbing peaks eluted from the column. Inthis case, results were recorded as % "peak B" (whole CAMPATH-1H(anti-CDw52 antibody).

The results are set out in the following Table 10.

                  TABLE 10                                                        ______________________________________                                        Added   % Peak B                                                              Cu (mM) PBS only  PBS + 2 mM EDTA                                                                              50 mM Citrate                                ______________________________________                                        0       42.92     100            100                                          1       21.47     98.95          94.71                                        2.5     18.72     36.96          94.66                                        5.0     0         0              93.43                                        7.5     0         0              92.82                                        10      0         0              92.57                                        12.5    0         0              84.85                                        15      0         0              32.53                                        20      0         0              15.48                                        ______________________________________                                    

Cleavage of CAMPATH-1H (anti-CDw52 antibody) in phosphate bufferedsaline alone at pH 7.2 is relatively rapid on incubation for 24 hours at62° C. even in the absence of added copper. In phosphate buffered salineplus 2 mM EDTA, pH7.2, cleavage is induced when greater than 1mM--copper is added. In 50 mM--citrate, pH 6.0, cleavage cakes placewhen copper in excess of 10 mM is added.

EXAMPLE 9

A similar experiment to Example 8 also investigated the effect ofvarying the pH. CAMPATH 1H (anti-CDw52 antibody) produced in CHO cellsof the type referred to in Example 1 in phosphate buffered saline, thebatch having no detectable copper as measured by atomic absorptionspectroscopy, was diluted 1:20 in phosphate buffered saline pH 7.2.Protein concentration was then determined as described in Example 8 andthe samples diluted to a protein concentration of 2 mg/ml with phosphatebuffered saline pH 7.2 or phosphate buffered saline pH 6.0 and the pHwas checked. Either 4 μl 0.1M˜trisodium titrate, pH 7.0 or 4 μl0.1M-EDTA, pH 7.0 was added to 200 μl aliquots of each of the CAMPATH-1H(anti-CDw52antibody) samples (2 mg/ml in phosphate bufferedsaline-either pH 7.2 or pH 6.0) to give a final concentration of about 2mM with respect to citrate or EDTA.

Copper was added up to 3 mM as 0 to 6 μl aliquots of 0.1M CUCl₂.2₂ O per200 μl CAMPATH-1H (anti-CDw52 antibody) (2 mg/ml) sample. 4 μl water wasadded to samples without copper. Samples were incubated at 62° C. for 24hours, centrifuged to remove any precipitated material and 50 μlaliquots analysed by size exclusion HPLC in the manner described inExample 8. Results, recorded as % "peak B" (whole CAMPATH-1H (anti-CDw52antibody)) are set out in the following Table 11.

                                      TABLE 11                                    __________________________________________________________________________            % Peak B                                                                      PBS only  PBS + 2 mM EDTA                                                                         BPS + 2 mM citrate                                Added Cu (mM)                                                                         pH 7.2                                                                             pH 6.0                                                                             pH 7.2                                                                             pH 6.0                                                                             pH 7.2                                                                             pH 6.0                                       __________________________________________________________________________    0       93.54                                                                              95.29                                                                              91.41                                                                              92.91                                                                              93.17                                                                              89.25                                        0.5     3.24 38.46                                                                              92.86                                                                              94.87                                                                              64.81                                                                              86.63                                        1.0     17.27                                                                              12.89                                                                              94.47                                                                              93.56                                                                              66.77                                                                              84.96                                        2.0     6.5  0    95.14                                                                              13   18.36                                                                              0.74                                         2.5     25   0    12.92                                                                              0    38.41                                                                              0.8                                          3.0     15.44                                                                              0    13.2 0    37.5 0.93                                         __________________________________________________________________________

The above table shows the approximate stoichiometry of binding of Cu²⁺by 2 mM-EDTA and 2 mM-citrate and the contributory effect of pH. 2mM-EDTA in phosphate buffered saline, pH 7.2, is the most effective insuppressing copper induced cleavage of Campath-1H. An approximate 1:1stoichiometry of binding is indicated at pH 7.2. Copper concentrationsin excess of 2 mM cause cleavage of CAMPATH-1H (anti-CDw52 antibody) in2 mM EDTA.

We claim:
 1. In an immunoglobulin composition of IgG₁ containing copperions in an amount sufficient to degrade the immunoglobulin, wherein theimprovement comprises the addition of an amount of a chelator of copperions sufficient to bind the copper ions present in the composition andprotect the immunoglobulin from degradation by the copper ions and thusstabilize the IgG₁ composition.
 2. A composition in accordance withclaim 1, wherein the immunoglobulin is a recombinant immunoglobulin. 3.A composition in accordance with claim 2, wherein the immunoglobulin isa chimetic or humanized immunoglobulin.
 4. A composition in accordancewith claim 2 or 3, wherein the immunoglobulin is produced from a Chinesehamster ovary (CHO) cell.
 5. A composition in accordance with claim 2 or3, wherein the immunoglobulin is produced from a myeloma cell.
 6. Acomposition in accordance with claim 1, 2 or 3, wherein theimmunoglobulin specifically binds to a tumor cell marker antigen.
 7. Acomposition in accordance with claim 1, 2 or 3, wherein theimmunoglobulin specifically binds to the CD2, CD3 CD4, CD5, CD7, CD8,CD11a, CD11b, CD18, CD19, CD25, CDw52, CD33 or CD54 antigen.
 8. Acomposition in accordance with claim 1, 2 or 3, wherein the chelator isethylenediamine tetraacetic acid (EDTA).
 9. A composition in accordancewith claim 8, wherein the amount of EDTA is 0.05 mM to 5 mM.
 10. Acomposition in accordance with claim 9, wherein the amount of EDTA is0.1 mM to 3 mM.
 11. A composition in accordance with claim 1, 2 or 3,wherein the chelator is citrate ion.
 12. A composition in accordancewith claim 11, wherein the citrate ion is alkali metal citrate.
 13. Acomposition in accordance with claim 1, 2 or 3, wherein the pH of thecomposition is within the range of 6 to 7.2.
 14. A composition inaccordance with claim 1, which is in the form of a liquid preparationsuitable for parenteral administration.
 15. A composition in accordancewith claim 1, which is in lyophilized form suitable for reconstitutioninto a liquid preparation suitable for parenteral administration.
 16. Astabilized immunoglobulin composition comprising an IgG₁ and copperions, wherein the copper is present in an amount sufficient to degradethe immunoglobulin, together with an amount of a chelator of copper ionssufficient to bind the copper ions present in the composition andprotect the immunoglobulin from degradation by the copper ions.